Defective regulation of gap junctional coupling in cystic fibrosis pancreatic duct cells

Abstract

The cystic fibrosis (CF) gene encodes a cAMP-gated Cl- channel (cystic fibrosis transmembrane conductance regulator [CFTR]) that mediates fluid transport across the luminal surfaces of a variety of epithelial cells. We have previously shown that gap junctional communication and Cl- secretion were concurrently regulated by cAMP in cells expressing CFTR. To determine whether intercellular communication and CFTR-dependent secretion are related, we have compared gap junctional coupling in a human pancreatic cell line harboring the DeltaF508 mutation in CFTR and in the same cell line in which the defect was corrected by transfection with wild-type CFTR. Both cell lines expressed connexin45 (Cx45), as evidenced by RT-PCR, immunocytochemistry, and dual patch-clamp recording. Exposure to agents that elevate intracellular cAMP or specifically activate protein kinase A evoked Cl- currents and markedly increased junctional conductance of CFTR-expressing pairs, but not in the parental cells. The latter effect, which was caused by an increase in single-channel activity but not in unitary conductance of Cx45 channels, was not prevented by exposing CFTR-expressing cells to a Cl- channel blocker. We conclude that expression of functional CFTR restored the cAMP-dependent regulation of junctional conductance in CF cells. Direct intercellular communication coordinates multicellular activity in tissues that are major targets of CF manifestations. Consequently, defective regulation of gap junction channels may contribute to the altered functions of tissues affected in CF.

Figure 1

Expression of CFTR and Cx45 in CFPAC-1 and PLJ-CFTR cells. (a) RT-PCR of mRNA isolated from both cell lines, as well as from T84, CAPAN, and SKHep1 cells, using primer pairs specific for CFTR (lanes 2–5) and Cx45 (lanes 6–10). Amplification products of the expected sizes for CFTR (expected size: 410 bp) were detected in T84 and PLJ-CFTR cells (lanes 1 and 4, respectively). No products were detected with these primers in CFPAC-1 and SKHep1 cells (lanes 3 and 5, respectively). mRNA for Cx45 (expected size: 309 bp) was detected in CAPAN, CFPAC-1, PLJ-CFTR, and SKHep1 cells (lanes 6, 8, 9, and 10, respectively), but not in T84 cells (lane 7). Molecular markers are shown in lanes 1 and 11 (b and c). Indirect immunofluorescence of CF and corrected pancreatic duct cells cultured on glass coverslips. Punctate labeling for Cx45 was detected in CFPAC-1 (b) and PLJ-CFTR (c) cell clusters at cell-cell contacts. Scale bar: 60 μm.

Figure 2

Differential activation of Cl^– currents by cAMP in CFPAC-1 and PLJ-CFTR cell pairs. Examples of membrane currents recorded from a CFPAC-1 (a) and PLJ-CFTR (b) cell pair. Whereas exposure of PLJ-CFTR cells to 10 μm forskolin, 500 μm 8-Br-cAMP, and 500 μm CPT-cAMP induced inward currents that could be blocked by 2 successive applications of 200 μM DPC (n = 6), the cAMP cocktail (cAMP) had no effect on membrane currents of CFPAC-1 cells. Note a small leakage current that developed with time in both traces. Bars indicate the duration of the drugs’ superfusion. The bath solution is renewed every minute. Dashed lines indicate the zero current level.

Figure 3

Differential effects of cAMP on junctional conductance of CFPAC-1 and PLJ-CFTR cell pairs. Examples of junctional conductances evaluated from a CFPAC-1 (a) and a PLJ-CFTR cell pair (b). Whereas exposure of PLJ-CFTR cells to 10 μM forskolin, 500 μM 8br-cAMP, and 500 μM CPT-cAMP increased their electrical coupling in a reversible manner, the cAMP cocktail (cAMP) was without effect on junctional conductance of the CFPAC-1 cell pair. Bars indicate the duration of drugs superfusion. Dashed lines indicate the zero junctional conductance level.

Figure 4

Distribution of junctional conductance values evaluated in CFPAC-1 and PLJ-CFTR cell pairs monitored under dual patch-clamp, with intracellular solutions supplemented or not with 20–100 μM S_p-cAMPS. As shown, S_p-cAMPS did not affect junctional conductance of CFPAC-1 cells. In contrast, junctional conductance values of PLJ-CFTR cells, which are lower than those measured in parental cells, are markedly increased (P < 0.05) in the presence of S_p-cAMPS in intracellular solutions. Bars represent the median values of junctional conductance.

Figure 5

Unitary conductance of the gap junction channels expressed in CFPAC-1 and PLJ-CFTR cells. (a) Example of gap junction channel activity in a CFPAC-1 cell pair that was monitored in the presence of halothane. Transitions of similar amplitudes but opposite polarities were recorded in both current traces (I₁ and I₂) during a 75-mV transjunctional potential. (b) Frequency distribution of transitions measured in CFPAC-1 (left) and PLJ-CFTR (right) cell pairs monitored under control conditions (open bars) or in the presence of S_p-cAMPS (filled bars). Both cell types showed 1 distribution of conductance values that could be described by Gaussian relation (solid lines). The latter distributions were not affected by 100 μm S_p-cAMPS added to the pipette solution.

Figure 6

Effects of cAMP on gap junction channel activity in a PLJ-CFTR cell pair. Traces represent junctional currents recorded under stationary conditions at a transjunctional potential of 55 mV. Four successive sweeps per experimental condition are shown. In the presence of 500 μm DPC, the channels remained closed almost all the time, with brief opening events (upward current deflections) being only occasionally detected. Addition of the cAMP cocktail (DPC + cAMP) increased the number of current transitions to the first and second levels of channel openings. Single-channel activity was again decreased when cAMP was omitted from the superfusing solution. Multiple levels of channel activity were rapidly detected as soon as DPC was washed out (Reversibility). Dashed lines represent the zero junctional current level.